WO1997039034A1 - Means for detecting bacteria of the taylorella equigenitalis species and their biological applications - Google Patents

Abstract

The invention concerns monoclonal antibodies and their biological applications. These monoclonal antibodies are characterised by the fact that they recognize an epitope of a bacterium of the T. equigenitalis species.

Description

MEANS FOR DETECTION OF TAYLORELLA EQUIGENITALIS BACTERIA AND BIOLOGICAL APPLICATIONS

The subject of the invention is means for the detection of bacteria of the genus Taylorella and their biological applications.

It relates in particular to the detection of T. equigenitalis and the treatment or prevention of infections caused by bacteria of this species. The first strain of T. equigenitalis was isolated by Crowhurst, 1977, Vet. Fairy. 100, 476 and characterized by Taylor et al. , 1978, Equine Vet. J. 10, 136-134. This bacterium is the agent of a venereal disease of equines called contagious equine metritis (hereinafter referred to as ERM).

Since the outbreak of this disease in 1977 in Newmarket (Great Britain), CEM has spread among the equine population in the world (Europe, USA, Japan). ECM was initially characterized by the appearance of purulent vaginal discharge caused by acute endometritis. The epidemiology and clinical manifestations of the disease have now changed. There are only a few rare foci with an acute form of CEM; these are then contaminations of several mares belonging to the same harem. The clinical forms of metritis have, indeed, become rare and T. equigenitalis is mainly found in asymptomatic carriers or in the pre-clinical stage. The disease is transmitted by stallions which show no clinical symptoms. The ECM constitutes an obstacle to the international exchange of equines and its detection is recommended by the OIE (Office International des Epizooties, list B). Indirect means of screening such as serology have been abandoned by many countries such as the USA, Great Britain and France.

Direct means of screening are practiced: screening by bacteriological culture in many countries, screening by indirect immunofluorescence. In France, prophylactic measures include both bacteriological culture and

1 indirect immunofluorescence (IIF).

Systematic screening of stallions has become compulsory prior to each breeding season. For both economic and organizational reasons, this systematic screening can only be done with one or two samples per animal and per season. The reliability of screening is therefore all the more crucial. The screening test for infection with T. equigenitalis currently carried out in France is mainly based on the isolation of the bacteria by culture on nutritive and / or selective media and on the identification of this agent according to morphological and biochemical criteria. However, T. equigenitalis is a very fragile and very slow growing bacterium (the observation time of the seed boxes is at least 6 days). It is, moreover, liable to be inhibited by other bacteria of the flora examined. The criteria for identifying the different strains of T. equigenitalis are themselves either too succinct and subject to variation (evidence of absence activity for the three classic enzymatic activities that T. equigenitalis presents), is too cumbersome to manage within the required time. Screening using only the bacteriological technique has therefore become a hazardous method of diagnosis. An undetermined percentage of healthy carriers is therefore considered to be uninfected each season.

A second screening test for infection with T. equigenitalis was selected in France. This test is based on the identification of the bacterium by indirect immunofluorescence using antiserum produced on rabbits and anti-rabbit fluorescent antibodies. This screening test has the advantage of delivering its results much faster (24 to 48 hours) than a test by bacteriological culture.

The use of this technique can however lead to excess errors (false positives), the antisera used giving rise, in many cases, to reactions with species other than T. equigenitalis.

The scope of these results is thus very limited: if the immunofluorescence test is negative, the approved laboratory can communicate a negative conclusion, but, if the result is positive, this result must be confirmed or invalidated by bacteriology.

The inventors have sought to remedy these difficulties in detecting infection with T. equigenitalis, by developing new means making it possible to identify a bacterium of the species T. equigenitalis without the risk of either false positives or false negatives. The The invention therefore aims to provide means for specific, highly reliable detection of T. equigenitalis, based on recognition of the defined antigen-antibody type. It also relates to the use of these means for the diagnosis, treatment and prophylaxis of diseases caused by T. equigenitalis.

According to a first aspect, the means of the invention are monoclonal antibodies characterized in that they recognize an epi tope of a bacterium of the species T. equigenitalis.

Advantageously, these antibodies do not exhibit cross-reactions with one or more epi topes of a Taylorella bacteria of a different species or of a bacteria of a different genus. They therefore make it possible to detect T. equigenitalis with certainty and, according to an aspect of great interest, using a single test.

The monoclonal antibodies of the invention (hereinafter abbreviated as mAb) are also as obtained from hybrids, by fusion of non-secreting murine myeloma cells with spleen cells from immunized mice using of a strain of the inactive species T. equigenitalis or of extract (s) of such a strain, cloning and selection according to the property of their culture supernatant to recognize one or more epi topes of a bacterium of the species T. equigenitalis, and recovery of the antibodies sought, followed where appropriate by their purification.

The invention also relates to the fragments of the mAbs defined above, more particularly their fragments Fv, Fab, F (ab ') 2. The mAbs of the invention and, where appropriate, their fragments, are further characterized in that they are capable of recognizing proteins of T. equigenitalis from the group comprising proteins such as the proteins of 150, 120, 52.7 or 22 (LPS) kDa.

According to a second aspect, the means of the invention are immunogenic proteins characterized in that they are capable of interacting with said mAbs or their fragments. These proteins are obtained, thanks to the so-called mAbs or their fragments, from T. equigenitalis, or by synthesis.

According to a third aspect, the means of the invention are anti -antibodies (hereinafter referred to as anti -AcM for short) and the fragments of these anti -anti co rps, these anti -AcM and their fragments being characterized in what they are capable of interacting with mAbs or their fragments defined above.

The invention also relates to methods for obtaining the means defined above.

To produce the mAbs of the invention, or the anti-mAbs, it is advantageous to use the technique of obtaining hydromes as described by Kôhler and Milstein in Nature 1975, 256, 495-497. The invention therefore provides a method for obtaining and selecting MAb defined above, characterized in that it comprises ^1: cell fusion of non-secreting murine myeloma with spleen cells from mice immunized using '' a strain of the species T. equigenitalis or extract (s) of such a strain, screening using a revelation technique, such as, in particular, indirect immunofluorescence, hybridomas whose culture supernatants react positively with a bacterium of the species T. equigenitalis or a fragment thereof this,

- the cloning of such hybridomas, with regard to their reactivity with respect to T. equigenitalis, and

- recovery of the mAbs sought, followed, where appropriate, by their purification. The invention also relates to the application of the above technique for the production of anti-mAb antibodies.

In this case, spleen cells from mice immunized beforehand using the mAbs already defined are used. The cloned strains can be stored in liquid nitrogen and their culture supernatants at -20 ° C. These strains which are characterized by the fact that they are capable of producing mAbs or respectively anti-mAbs, as defined above, also fall within the scope of the invention. In general, the invention relates to strains of hybridomas as obtained according to the methods defined above.

The mAb fragments and the anti-mAb can be easily obtained using conventional enzymatic techniques.

With the three aspects defined above, namely the mAbs or their fragments, the immunogenic proteins, and the anti-mAbs or their fragments, the invention provides the means for establishing, either directly or indirectly, possible contamination of a sample or culture with a bacterium of the species of T. equigenitalis.

In the context of such a determination, the invention relates to a method of identifying a bacterium of the species T. equigenitalis or of one or more epi topes of such a bacterium in a sample or in a culture, characterized in that it comprises:

- bringing the sample or the culture to be analyzed, likely to contain T. equigenitalis, into contact with i. an effective amount of at least one mAb or a fragment of mAb as defined above and, optionally, blocking of the non-antigen-antibody reactions, for example by saturation of the sample or of the culture to be analyzed with using serum, such as mouse serum, lacking anti-T antibodies. equigenitalis, ii. or alternatively, to demonstrate the presence of antibodies directed against T. equigenitalis, with an effective amount of an immunogenic protein or of anti-mAb antibodies, or of fragments of the latter, as defined below. us, under conditions allowing a reaction of the type anti gene-anti co rps, and the revelation of the reaction product of type antigen-antibody possibly formed.

The contacting step is carried out under conditions in particular of duration, temperature, buffer, allowing the establishment of an antigen-antibody type reaction. For the revelation, markers are used, for example fluorescent, enzymatic, radioactive or luminescent markers. It will be noted that the judicious choice of a particular mAb, or of a fragment of this mAb, makes it possible to directly identify a given epi tope of T. equigenitalis in a sample or a culture to be analyzed. By using an immunogenic protein or an anti-mAb antibody or a fragment of the latter, a prior contact of the sample or of the culture with the bacteria will be demonstrated.

The absence of cross-reactions of the mAbs of the invention and their fragments with epithets of bacteria of the genus Taylorella other than T. equigenitalis, and bacteria of a different genus, is advantageously taken advantage of for the diagnosis of pathologies linked to T. equigenitalis. The invention therefore also relates to the use of said mAbs and their fragments for the diagnosis of an infection with T. equigenitalis, more particularly contagious equine metritis, characterized in that it comprises: - bringing into contact one or more mAbs of the invention, or their fragments, with a biological sample, and

- revelation of the antigen-antibody type reaction produced in the case of the presence of T. equigenitalis in the sample,

- And, optionally, the blocking of non-antigen-antibody reactions, for example, by saturation of the sample taken using a serum, such as a mouse serum, devoid of anti-T antibodies. equigenitalis. The contacting and revelation steps are advantageously carried out as indicated for the preceding method. The invention also provides kits for implementing the identification methods and diagnostic methods described above.

These kits are characterized in that they contain

one or more mAbs or their fragments or at least one immunogenic protein, or one or more anti-mAbs or their fragments,

the reagents, in particular the markers or buffers, allowing the revelation of the targeted immunological reaction, and, optionally, reagents for blocking non-antigen-antibody reactions such as mouse serum,

- as well as instructions for use. According to another advantageous arrangement of the invention, the mAbs and their fragments defined above can be used in therapy to combat infection by T. equigenitalis, and more particularly against contagious equine metritis. The invention thus also relates to pharmaceutical compositions containing one or more mAbs, or their fragments, defined above, as drug vectors or as passive immunotherapy agents, alone or in combination with pharmaceutically inert vehicles. It also relates to their use for the development of biosensors.

According to yet another provision, the invention relates to the use of immunogenic proteins and anti-mAbs or their fragments for the preparation of vaccine compositions preventive of infection by T. equigenitalis. The vaccine compositions of the invention are characterized in that they contain at least one immunogenic protein or an anti-mAb or their fragments, as defined above, in an amount sufficient to elicit an immune response, in association with excipients physiologically acceptable.

Other characteristics and advantages of the invention will be given in the examples which follow. In these examples, reference is made to FIGS. 1 to 3, which respectively represent:

FIG. 1 represents a photo of an IIF (indirect immunofluorescence) test on T. equigenitalis in the presence of mAb according to the invention,

FIG. 2, a photo of an immunoblot after reaction of T. equigenitalis proteins with mAbs of the invention and serum of immunized mice (positive serum),

FIG. 3, a photo of a dot blot produced on the undenatured proteins of a reference strain of T. equigenitalis and incubated with the mAbs according to the invention, a positive mouse serum (SP) or a negative mouse serum (SN) (non-immunized mouse).

Example 1: Obtaining and selecting hybridomas capable of producing anti-T monoclonal antibodies. equigenitalis strains of T. equigenitalis used for immunization

The results obtained with the following nine strains are reported: - two reference strains (Rl-16 and R2-19), from the National Center for Veterinary and Food Studies - Central Laboratory for Veterinary Research CNEVA-LCRV, Maisons -Al fort, France,

- seven strains called wild strains isolated in four different regions of the

North West of France (Indre et Loire, Calvados, Côtes d'Armor and Orne).

Ces souches sont identifiées dans le tableau I ci- après

These strains are identified in Table I below

TAELEAU I

Designation of Sources Resistance to the Streptomycin strain

Rl-16/16 CNEVA S R2-19 / 19 CNEVA R

1 / 129S LVD37 R

2/1 LVD14 R

3 / 12.397 LDA22 R

4 / 26.658 LDA22 R 5 / 7001-01 LDA22 R

6/250 LVD61 R

7/715 LVD61 R

S = sensitive R = resistant

All these strains are grown on chocolate agar agars with or without the addition of actidione and streptomycin. They are incubated in a humid atmosphere at 7% C0 ₂ . The enzymatic reaction and sugar fermentation analyzes are carried out using the API-NH system (ELoMérieux, Marcy-1 * Etoile, France).

In addition, these strains are tested for their catalase, cytochrome oxidase activity and by the serum agglutination test (SAT), using a polyclonal rabbit antiserum.

Most of them have - a Gram-negative cocobacillary form,

- a catalase and cytochrome oxidas e activity, and they respond positively to the SAT agglutination test. It is found that they all exhibit a positive alkaline phosphatase and gamma glutamyl transferase activity (except the strain of field 5 which exhibits a negative gamma glutamyl transferase activity), - penicillinase, ornithine-decarboxylase, urease, lipase, beta-galactosidase and proline- activities negative amylase. We also note that they do not metabolize sugars (glucose, fructose, maltose, sucrose).

In addition, they have very similar polypeptide and lipopolysaccharide profiles.

The two reference strains RI-16 and R2-19 therefore exhibit the properties generally observed for all the strains of T. equigenitalis studied in the prior art and are therefore used for the immunization of mice.

- mouse immunization

The reference strains Rl-16 and R2-19 are washed twice in 0.1 M PBS buffer, pH 7.4, and inactivated by heating at 56 ° C for 75 min. The cells are then diluted in PBS, until bacterial suspensions of optical density 0.77 to 380 nm. They are then divided into aliquots and stored at -80 ° C until use.

Adult BALB C mice are injected 0.5 ml of Rl-16 and R2-19 bacterial suspension emulsified with the complete Freund's adjuvant (2 mice per strain) into BALB C adult mice. A booster injection is given on the 14th day with the same preparation. On the 21st day, the mice are immunized with 0.2 ml of suspension without adjuvant by the intravenous route and the spleen cells are collected 2 days later.

- production of hybridomas

Hybridomas are produced according to the standard procedure described by Kohler and Milstein (see reference below).

SP2-0-Agl4 mouse myeloma cells and immune spleen cells are fused in a 1/5 ratio using PEG 1500 (Sigma, Isle d'Abeau, France) and maintained in cell culture plates at 96 wells containing mouse macrophages or spleen feeder cells or an OPI supplement (Sigma) in a selective HAT-DMEM medium.

Hybridoma growth is observed in 820 of the 1020 wells used (81.37%). IIF tests are carried out on 60 of these 820 wells to detect hybridomas producing the desired monoclonal antibodies. screening of hybridomas and monoclonal antibodies produced

Hybridomas are tested by indirect immunofluorescence (IIF) for the ability of their supernatants to recognize the two reference strains of T. equigenitalis. The standard procedure described by Vaissaire et al. (1992), Bail. Acad. Vet. Fr. 65,

161-170. After two washes in 0.1 M PBS, pH 7.4, the bacterial strains are resuspended in PBS buffer containing, in addition, 1% formaldehyde in order to obtain a suspension having a turbidity of 1 in the scale by Mac Farland. 10 μl of this suspension are applied to each spot of fluorescent strips.

After drying for 15 min at 37 ° C., the strips are fixed in pure acetone for 15 min at room temperature. After drying, the slides are incubated with 40 μl of hybridoma supernatants, for 30 min at

37 ° C.

The slides are then washed in a P BS bath with stirring for 15 min. After rinsing in distilled water and drying, the slides are incubated for 30 min at 37 ° C. with 40 μl of a solution of ceine fluorinated isothiocyanate conjugated to fraction F (ab) 2 of anti-mouse rabbit (Eurobio Les Ulis, France), diluted 1/40 in

PBS containing Evans blue (1/10000). The coverslips are finally washed in PBS, rinsed in distilled water, dried as indicated above, mounted in PBS containing 1% glycerin and examined using a fluorescence microscope.

Unimmunized mouse serum is used as a negative control. The FITC mouse antiserum conjugate is incubated with each bacterial strain to serve as a conjugate control.

Clones positive for the IIF test are transferred for expansion before cloning into 24-well plates containing the HAT-DMEM medium. An IIF test is reported in FIG. 1 on T. equigenitalis in the presence of mAb according to the invention. This figure shows a strong fluorescence of the bacterial wall.

4 to 7 days later, the hybridomas of these wells are cloned by the limit dilution method in order to obtain a single cell per well in a 96-well tissue culture plate using the HT-DMEM medium and feeder cells. Single clone wells are screened with IIF and positive cells are frozen in liquid nitrogen.

Among all the positive clones 14 of them are used for the production of monoclonal antibodies and the characterization of these antibodies.

The tissue culture supernatants of hybridomas are buffered by the addition of Tris IM, pH 8.0 (vol. 1/20) and sodium azide (0.02%). Aliquots are made and stored at -20 ° C.

Example 2: Characterization of the anti-T monoclonal antibodies. equigenitalis

- specificity of monoclonal antibodies In order to verify the specificity of the monoclonal antibodies, the supernatants of the 14 hybridoma clones obtained according to Example 1 are tested by IIF according to the capacity of their supernatants to recognize other bacterial strains than the two reference strains R-16 and R-19 used for immunization, namely: the 7 wild strains of T. equigenitalis described in Example 1, and - bacterial strains described in the prior art as giving rise to cross-reactions with the T antisera equigenitalis or commonly present in the genital flora: Actinobacillus equuli, Pseudomonas aeruginosa, Pasteurella multocida, Pasteurella haemolytica, Streptococcus equi,

Staphyloccoccus aureus, Pseudomonas fluorescens and Klebsiella pneumoniae. These bacteria are grown on a Columbia-based blood-agar medium.

The results obtained are reported in Table II below.

Les 14 anticorps monoclonaux testés reconnaissent les sept souches sauvages de T. equigenitalis. 3 d' entre eux donnent une réponse plus faiblement positive, à savoir 7 B7.1 ; 7 B7.10 et 10C9.6. Aucun des 14 anticorps monoclonaux testés ne reconnaît une des 8 souches bactériennes qui n' appartiennent pas à l'espèce T. equigenitalis.

The 14 monoclonal antibodies tested recognize the seven wild strains of T. equigenitalis. 3 of them give a weaker positive response, namely 7 B7.1; 7 B7.10 and 10C9.6. None of the 14 monoclonal antibodies tested recognizes one of the 8 bacterial strains which do not belong to the species T. equigenitalis.

These results demonstrate the specificity of the 14 monoclonal antibodies tested against the strains of T. equigenitalis and the absence of cross-reactivity between T. equigenitalis and other bacteria, not belonging to the species T. equigenitalis, and either having have been described with the tools of the prior art as having a cross reactivity with this species (Actinobacillus equuli, Pasteurella multocida, Pasteurella haemolytica, Straphylococcus aureus, Pseudomonas fluorescens) either forming part of the current genital flora [Streptococcus equi, Klebsiella pneumoniae, Pseudomon aeruginosa). The positive reactions of the rabbit polyclonal antiserum observed in IIF with Staphylococcus aureus and Pseudomonas fluorescens were therefore not observed with the monoclonal antibodies of the invention.

The monoclonal antibodies object of the present application do not detect an antigenic difference between the different strains of T. equigenitalis tested.

- SAT (Agglutination Test Serum)

To test the reactivity of monoclonal antibodies to SAT, only the strain R-19 was used. The results obtained are given in column 4 of Table III below.

13 of the 14 monoclonal antibodies give a positive response.

- localisation d'épitopes spécifiques

- localization of specific epitopes

preparation of protein and lipopolysaccharide extracts of strain R-19 from r. equigenitalis

- Extract in non-denaturing conditions (EN) of T. equigenitalis

The cells of T. equigenitalis were harvested by centrifugation (6000 g, 10 min) and washed three times in a 0.1 M PBS solution at pH 7.4. The pellets were resuspended in a small volume of SDS buffer (2% sodiumdodecyl sulfate, PBS pH = 7.4) and incubated at 37 ° C for 30 min. As a result of this process, proteins retain their biological activity. After extraction in SDS buffer, the integrity of the cells was checked by observations using phase contrast microscopy. After centrifugation (10,000 g, 10 min), the supernatants containing EN were completely dialyzed against distilled water at 4 ° C for 48 h, divided into aliquots and kept frozen (-80 ° C) until to use. The protein concentration of EN was determined using the BLoBad protein test (BloRad, Ivry-sur-Seine, France).

- Extract in denaturing conditions ED

The EN extracts of the T. equigenitalis strains were dissolved in a sample solvent (Tris.HCl 0.1 M pH 6.8; glycerol 10%; SDS 2%; β-mercaptoethanol 2 mM and bromophenol blue 0.01% ) in order to obtain a protein concentration of 1 mg / ml, then were brought to a boil at 100 ° C for 5 min (extract under denaturing conditions of T. equigenitalis, ED).

- Lipopolysaccharide extract (LPS) EN extracts digested with proteinase K were used as LPS extracts (Hanner et al., 1991 Am. J. Vet. Res. 52,1065-1068). 10 μl of EN were diluted in 35 μl of LPS digestion buffer. This LPS digestion buffer consists of 0.0625 M Tris.HCl pH 6.8; 0.1% SDS; 10% glycerol and 5 μg of proteinase-K (Sigma). These preparations were incubated at 57 ° C for 1 hour and heated to 100 ° C for 5 min before electrophoresis.

- electrophoresis on sodium dodecyl sulfate polyacrylamide gel (SDS - PAGE)

For the separation of bacterial proteins, a discontinuous SDS-PAGE electrophoresis was used (Laemmli, 1970, Nature, 227, 680-685). The separation gel contained 12% acrylamide and the s taking gel 4% acrylamide. 20 μl of each ED sample were deposited at the bottom of the wells at a concentration equivalent to 5 μg of proteins per lane. The electrophoresis was carried out at 100 V, 50 mA (direct current) for 10 h in a vertical unit of gel plates (Hoefer Scientific Instr., San Francisco, CA). For molecular weight determinations, a kit intended for the calibration of low molecular weights (Pharmacia-BLotech, Saint-Quentin en Yvelines, France) was used. To visualize the bands on the polyacrylamide matrix, we used Coomasie staining R350 (Pharmacia-BLotech, France) and for the visualization of the LPS components, the silver coloration (Tsai and Frasch, 1982 Anal. ELochem. 199, 115-119).

- immunoblotting

The protein bands were transferred from the gel onto an Immobilon ^R PVDF membrane (Millipore Corp., St Quentin en Yvelines, France) by electroblotting using an electrophoretic transfer cell MiniTrans- Blot ^R (BLoRad) with a transfer buffer solution (Tris 25 mM; glycine 192 mM; methanol 20% v / v; pH = 8.3) at 100 V, 250 mA for 1 hour. To verify the conditions of electrophoretic transfer and to identify the protein bands on the membranes, the coloration of the total proteins by colloidal gold (BLoRad, Colloidal Gold Total Protein Stain) was used. After transfer, the membranes were immersed for 30 min in a blocking solution (3% gelatin in 20 mM Tris and 0.5 M NaCl) and rinsed with gentle stirring in a washing solution (20 mM Tris; NaCl, 0.5 M; Tween ^R 20 0.05%). The membranes were then brought into contact with monoclonal antibody solutions diluted from 1/100 to 1/1000 in the antibody buffer (Tris 20 mM; 0.5 M NaCl, Tween ^R 20 0.05% gelatin 1% ) for 180 min at 25 ° C. The binding of monoclonal antibodies to the peptide bands was visualized using alkaline phosphatases (AP) conjugated with IgG immunoglobulins. goat (heavy and light chains) anti-mice (BLoRad, dilution to 1/2000) and using a substrate solution for PA (EoRad).

A positive serum from mice immunized with a reference strain of T. equigenitalis and a negative serum from non-immunized mice were used as experimental controls. FIG. 2 illustrates an immunoblot between the bacterial proteins and the mAbs according to the invention on the one hand and the positive serum of mice on the other hand.

The positive serum collected from immunized mice reacts with 5 proteins of the strain R-19: 120 kDA; 52.7 kDA; 33.4kDA; 17.5 kDA and 22 (LPS) kDA.

8 of the 14 monoclonal antibodies tested react positively and 6 of them negatively. The specific epi topes recognized by these 8 positively reactive monoclonal antibodies are:

150 kDa for the monoclonal antibody 3B6.1, 120 kDa for the monoclonal antibody 11C9.1, 52.7 kDa for the monoclonal antibodies 7B8.1 and

7C4.10,

22 kDa (LPS) for monoclonal antibodies 7B7.10, 7D7.3, 11C9.4 and 11C9.5

These results are also collated in Table III, columns 5 and 8.

- dot-blotting

Immobilon ^R PVDF (Sigma) membranes were pre-wetted with 100% methanol solution for

1 to 3s, immersed in distilled water for 1-2 min to elute the methanol and balanced in a solution washing (Tris 20 mM; NaCl 500 mM; Tween ^R 20 0.05%; pH = 7.5). The extracts EN and ED were fixed to the membranes by incubation for 1 hour at room temperature. The dot membranes were washed twice for 10 min in the washing solution and then immersed in the blocking solution (3% gelatin in 20 mM Tris and 500 mM NaCl) for 1 hour. The membranes were washed twice as above and incubated with the selected monoclonal antibodies under the same conditions as for immunoblotting.

The binding of the monoclonal antibodies to the dot blot membranes was revealed using PA conjugated to goat IgG immunoglobulins (heavy and light chains) anti-mice and using a substrate solution for PA (BLoRad) .

The same sera, positive and negative controls, were used as for the immunoblotting.

To determine whether the negative results observed on immunoblotting are due to the fact that the epi topes were damaged by the denaturing reagents used to prepare the extracts, the 14 monoclonal antibodies are compared in dot-blot with the extracts EN and ED of the strain R-19.

In FIG. 3, the R19 proteins which reacted on tracks 1 to 14 with the mAbs of Table III, on the SP track with the positive mouse serum and on the SN track with the negative mouse serum are reported in dot blot. . The results obtained are also collated in Table III, columns 6 and 7. The 6 antibodies which exhibit a negative immunoblot also exhibit a negative dot-blot with the denatured extracts of the strain R-19 (Table III, columns 5 and 6). However, they show a positive dot-blot with the non-denatured extracts (Table III, column 7).

Under non-denaturing conditions (treatment with SDS only), the conformation and activity of the proteins remain intact but, under reducing conditions (treatment with β-mercaptoethanol and high temperatures), the conformation of certain proteins changes and the epi topes are destroyed. . The absence of reactivity of the 6 monoclonal antibodies tested in immunoblotting with the R-19 strain is therefore very probably due to such changes of conformation and destruction of epithodes.

8 monoclonal antibodies which retain their reactivity on ED bacterial extracts were therefore produced.

These 8 monoclonal antibodies can therefore constitute reagents suitable for the detection of antigens of T. equigenitalis and, more particularly, for the diagnosis of CEM. Such antibodies can be used to characterize bacteria of the Taylorella genus in any biological preparation using denaturing conditions.

- Determination of the isotype

For the determination of the isotype of the monoclonal antibodies, the immunotype kit from Sigma was used which consists of strips of ni trocellulose pre¬ coated with anti-isotype antibodies of mouse immunoglobulins. After additional incubations, the identity The results obtained appear in column 9 of Table III.

The 14 monoclonal antibodies produced are part of IgM for 5 of them, IgG2b for 4 of them, IgG3 for 3 of them and IgGl for 2 of them.

Example 3:

Comparative test of the various diagnostic tests for ECM

a) bacteriological culture of the bacterial flora b) detection by polyclonal and IIF c) detection by the invention object of the present application: monoclonal and IIF.

During 1 month, 368 swabs of mares (clitoral fossa, cervix) and stallions (pre-ejaculatory liquid, urethral fossa) were studied by the two immunofluoric techniques, the technique in the sense of the memorandum from the Ministry of Agriculture and Fisheries (DGAL / SDSPA / N95 / N ° 8037) with polyclonal antibodies and the technique according to the invention. The positives by one of the two techniques were isolated by culture on agar media. 64 samples were found positive with polyclonal antibodies and 17 with monoclonal antibodies; no culture allowed isolating T. equigenitalis bacteria. These results clearly demonstrate the strongest specificity provided by the invention in this study. Example 4: Another comparative test

A second trial aimed at comparing the screening for ECM by bacteriological culture, by polyclonal and IIF and by the invention which is the subject of the present application (monoclonals and IIF) was carried out on 1014 samples representing all the requests for analysis. 1 T. equigenitalis was isolated by bacteriological culture (out of 1014 samples), 58 fluorescences were established with the monoclonal antibodies according to the invention (6%) and 409 with polyclonal antibodies (40%).

The differences measured between monoclonal and polyclonal antibodies are statistically significant with a probability greater than 99.9% (Chi-square test).

Both antibody and indirect immunofluorescence screening techniques, namely the "polyclonal antibody" technique and the technique which is the subject of the present invention, have both screened for T. equigenitalis isolated by bacteriological culture.

The specificity of the monoclonal antibodies according to the invention, used in the context of indirect immunofluorescence, is better than that of polyclonal antibodies (94% vs 60%).

Example 5: Elimination of non "antigen-antibody" reactions.

Nonspecific reactions can sometimes be obtained between antibodies and Staphylococcus protein intermediary (protein A for S. aureus and protein G for Streptococci of groups C and G). The reactions are not of the antigen-antibody type.

Such non-specific reactions can be observed with the monoclonal antibodies according to the invention: in fact, 2 strains of bacteria known to produce proteins A and G (Staphylococcus aureus strain Cowan and Streptococci strain 26RP66) were subjected to the detection technique according to the invention, namely monoclonal antibodies and indirect immunofluorescence, and both gave fluorescence (the strain R-19 of T. equigenitalis served as an experimental control).

In order to eliminate these non-specific reactions, a so-called blocking technique has been developed.

Monoclonal antibodies according to the invention conjugated to FITC, intended for detection by direct immunofluorescence, have been produced.

Two monoclonal antibodies according to the invention, an IgG2b (10C9.6) and an IgG3 (7C4.10) were concentrated 10 times by ammonium sulfate precipitation and purified on a column of Protein A Sepharose (Pharmacia) by adsorption in a Tris lOOmM buffer pH 8 and elution in a lOOmM glycine buffer pH 3. The antibodies thus purified were labeled with FITC Isomer gamma

(fluorescein isothiocyanate) and antibody conjugates-

FITC were separated from the unlabeled molecules by passage through a Sephadex G25 column (Pharmacia).

Three types of blades were produced:

T. equigenitalis strain R-19 resistant streptomycin, - Staphylococcus aureus Cowan strain,

- Group C Streptococcus strain 26RP66.

These slides were then subjected to blocking by incubation at 37 ° C for 1 h in a serum devoid of anti-T antibodies. equigenitalis. Three sera were compared: mouse serum, rabbit serum and human serum.

After washing with PBS and rinsing with distilled water, the slides are incubated for 1 hour at 37 ° C. with the FITC-labeled monoclonal antibodies according to the invention described above.

After washing and final rinsing, the slides are mounted in glycerin, buffered and examined under a fluorescence microscope. These three blocking techniques give fluorescence for the T. equigenitalis slides and do not give fluorescence for the non-specific binding slides (S. aureus and Streptococcus).

The best blocking was obtained with mouse serum.

It is therefore possible with the detection technique according to the present invention to eliminate non-specific reactions while retaining the specific anti gene-an ti co rps reaction. This serum blocking technique devoid of anti-T antibodies. equigenitalis and direct immunofluorescence can be advantageously used in confirmation of the positive results obtained by the technique of indirect immunofluorescence and monoclonal antibodies according to the invention. Example 6: Production of anti-Taylorella equigenitalis antibodies.

1. Production of anti-T monoclonal antibodies. equigenitalis (AcMl)

We operate as indicated above.

2. Purification of mAbs

The mAbs are precipitated by adding saturated ammonium sulfate to the final concentration of 50%. After centrifugation, the precipitate is resuspended in PBS, then filtered through Sephadex® G75 gel (Pharmacia) and finally purified by affinity chromatography on a column of protein A- Sepharose® CL-4B

3. Preparation of the immunogen The purified mAbs are homopolymerized in the presence of 0.25% glutaraldehyde for hours at 4 ° C. The reaction is stopped by adding a 0.2M glycine buffer and the polymers are dialyzed against PBS.

4. Immunization of mice BUJEV'C mice are immunized by 1 SC injection of a mixture of equal parts of 50 μg of polymerized mAb and complete Freund's adjuvant. 2 subsequent injections are given 2 weeks apart, one with incomplete Freund's adjuvant, and the other without any adjuvant and peritoneally.

5. Obtaining anti-body monoclonal antibodies against T. equigenitalis. (AcM2)

We proceed as described above.

6. Purification of the AcM1 Fab Fragments Fab fragments of the AcM1 antibodies are purified after digestion of the AcM1s with papalin (incubation 45 min at 37 ° C mAbs in a solution of papain, 2-β-mercaptoethanol, EDTA 1.5M at pH8. the ratio is 10 μg of papain per mg of mAb. Digestion is stopped by the addition of 10MM N-methylmaleimide (Sigma). The undigested antibodies and the Fc fragments are eliminated by affinity chromatography on a column of protein A-Sepharose CL-4 E® (Pharmacia). The purity of the Fab fragments is verified by SDS-PAGE.

7. Screening of AcM2 Producing Hybridomas by an ELISA Test

The microplates (Maxisorb, Nunc) are incubated for 16 h at 4 ° C with 100 μl / well of a suspension of 0.2 μg / ml of Fab in carbonate buffer pH8. The microplates are washed 3 times with P BS-Tween 20® (0.05%), pH 7.2, then the non-specific sites are blocked by a solution of BSA 2% in P BS-Tween 20® for 30 min at 37 ° C. After 3 washes with PBS-Tween 20®, the culture supernatants of the hybridomas are incubated 1 H at 37 ° C. After 3 washes with P BS-Tween 20®, the reaction is revealed by an anti-mouse conjugate labeled with peroxidase and its substrate.

Hybridomas positive by the ELISA test are selected and the supernatants are used for the preparation of the vaccine. 8. Preparation of the vaccine

The AcM2 antibodies of the selected hybridomas, then their corresponding Fab fragments are purified according to the methods described below. The Fab fragments are coupled to the limpet hemocyanin keyhole (KLH, Sigma) by incubation for 16 h at 4 ° C. in a 0.05% glutaraldehyde (Sigma) solution, in a ratio of 1/1. The reaction is stopped with a 0.02M glycine solution and the conjugates are dialyzed against PBS. The protein is dosed at 25-100 μg per dose of vaccine and the vaccine is supplemented with alumina hydroxide as an adjuvant.

Claims

1 / Monoclonal antibodies or their fragments, more particularly, their fragments Fv, Fab, F (ab ') 2, characterized in that they recognize an epi tope of a bacterium of the species T. equigenitalis. 2 / Monoclonal antibodies or their fragments, more particularly their fragments Fv, Fab, F (ab ') 2, according to claim 1, characterized in that they do not exhibit cross-reaction with one or more epi tope (s) d '' a bacteria of a different Taylorella species or a bacteria of a different genus. 3 / Monoclonal antibodies or their fragments, according to claim 1 or 2, characterized in that they are capable of recognizing proteins of T. equigenitalis from the group comprising proteins such as proteins of 150 kDa, 120 kDa, 52.7 kDa or 22 (LPS) kDa. 4 / Monoclonal antibodies, characterized in that they can be obtained from hybrids - by fusion of non-secretory murine myeloma cells with spleen cells from immunized mice using a strain of species T inactivated equigenitalis or extract (s) of such a strain, and - cloning and selection according to the property of their culture supernatant to recognize one or more epi topes of a bacterium of the species T. equigenitalis , - recovery of the desired monoclonal antibodies, followed if necessary by purification. 5 / Immunogenic proteins, characterized in that they are capable of interacting with antibodies monoclonals or their fragments according to any one of claims 1 to 4. 6 / Monoclonal antibodies, and their fragments, particularly their Fv, Fab, F (ab ') 2 fragments, characterized in that they are anti-anti-bodies, namely antibodies capable of interacting with monoclonal antibodies or their fragments according to any one of claims 1 to 4. 7 / A method for obtaining monoclonal antibodies according to any one of claims 1 to 4, characterized in that it comprises: the fusion of non-secretory murine myeloma cells with spleen cells from immunized mice using of a strain of the species T. equigenitalis or of extract (s) of such a strain, screening using a revelation technique, such as in particular indirect immunofluorescence, of hybridomas including the supernatants of culture show a positive reaction with a bacterium of the species T. equigenitalis or a fragment thereof, the selection by cloning of such hybridomas with regard to their reactivity, with respect to T equigenitalis, and - the recovery of monoclonal antibodies, followed if necessary by their purification. 8 / A method for obtaining monoclonal antibodies according to claim 6, characterized in that it comprises: the fusion of non-secretory murine myeloma cells with spleen cells from immunized mice using monoclonal antibodies or their fragments as defined in one of claims 1 to 4, screening using a revelation technique, such as in particular indirect immunofluorescence, of hybridomas in which the culture supernatants exhibit a positive reaction with one of said monoclonal antibodies or their fragments, the selection by cloning of such hybridomas, and - the recovery of anti-anti body research. 9 / Hybridoma strains characterized in that they are capable of secreting monoclonal antibodies according to any one of claims 1 to 4. 10 / Hybridoma strains characterized in that they are capable of secreting monoclonal antibodies according to claim 6. 11 / Method for identifying a bacterium of the species T. eguigemtaiis in a sample or in a culture, comprising: - bringing the sample or the culture to be analyzed, likely to contain T. equigenitalis, into contact with an effective amount of at least one monoclonal antibody or a fragment of such an antibody according to any one of the claims 1 to 4 and, optionally, blocking of non-antigen-antibody reactions, ii. or, alternatively, to demonstrate the presence of antibodies directed against T. equigenitalis with an immunogenic protein according to claim 5 or an antibody according to claim 6, under conditions allowing a reaction of the anti gene-antico rps type, and - the revelation of the reaction product of antigen-antibody type possibly formed. 12 / Method for diagnosing an infection with T. equigenitalis, more particularly contagious equine metritis in a sample or a culture, comprising: - bringing into contact one or more monoclonal antibodies according to any one of claims 1 to 4 or their fragments, with a biological sample, and - revelation of the antigen-antibody type reaction produced in the case of the presence of T. eguigenitalis in the sample, - And, optionally, blocking of non-antigen-antibody reactions, for example, by saturation of the sample taken using a serum devoid of anti-T antibodies. equigenitalis. 13 / Kits for the implementation of a method according to one of claims 11 or 12, characterized in that they comprise - one or more monoclonal antibodies, or their fragments, according to any one of claims 1 to 4, or at least one immunogenic protein according to claim 5, or one or more monoclonal antibodies, or their fragments, according to claim 6, - the reagents, in particular the markers or buffers, enabling the targeted immunological reaction to be carried out, and, optionally, reagents for blocking non-antigen-antibody reactions such as mouse serum, - as well as instructions for use. 14 / Pharmaceutical compositions, characterized in that they contain one or more antibodies monoclonals, or fragments thereof, according to any one of claims 1 to 4, as drug vectors or as passive immunotherapy agents, alone or in combination with pharmaceutically inert vehicles. 15 / Vaccine compositions, characterized in that they contain, in association with physiologically acceptable excipients, at least one immunogenic protein as defined according to claim 5, or an antibody according to claim 6, or a fragment of such an antibody , in sufficient quantity to elicit an immune reaction. 16 / Use of the monoclonal antibodies according to one of claims 1 to 4 for the development of biosensors.